CRISPR-Cas12a induced DNA double-strand breaks are repaired by locus-dependent and error-prone pathways in a fungal pathogen

Abstract

AbstractCRISPR-Cas mediated genome engineering has revolutionized functional genomics. However, basic questions remain regarding the mechanisms of DNA repair following Cas-mediated DNA cleavage. We developed CRISPR-Cas12a ribonucleoprotein genome editing in the fungal plant pathogen, Magnaporthe oryzae, and found frequent donor DNA integration despite the absence of long sequence homology. Interestingly, genotyping from hundreds of transformants showed that frequent non-canonical DNA repair outcomes predominated the recovered genome edited strains. Detailed analysis using sanger and nanopore long-read sequencing revealed five classes of DNA repair mutations, including single donor DNA insertions, concatemer donor DNA insertions, large DNA deletions, deletions plus donor DNA insertions, and infrequently we observed INDELs. Our results show that different error-prone DNA repair pathways resolved the Cas12a-mediated double-strand breaks (DSBs) based on the DNA sequence of edited strains. Furthermore, we found that the frequency of the different DNA repair outcomes varied across the genome, with some tested loci resulting in more frequent large-scale mutations. These results suggest that DNA repair pathways provide preferential repair across the genome that could create biased genome variation, which has significant implications for genome engineering and the genome evolution in natural populations.